Stabilization and acoustic activation of polymeric micelles for drug delivery

ABSTRACT

Methods are disclosed in which a micelle is stabilized against degradation upon dilution. The micelle comprises molecules of a block polymer having a hydrophobic block and a hydrophilic block. The hydrophobic block forms a core of the micelle with corona from the hydrophilic block. The methods for stabilizing the core are (1) by chemically cross-linking, (2) incorporating a hydrophobic oil (vegetable oil) in the core to render it more hydrophobic and stable, and (3) incorporating a cross-linked interpenetrating network of a stimuli-responsive hydrogel into the core. The hydrogel is responsive to any stimuli, but preferably temperature or pH. A substance such as, drugs, can be incorporated into the dense inner core of the micelles. 
     When subjected to ultrasound, the micelles release the substance, and then reversibly revert to a stable dense core and re-encapsulating the substance when the ultrasound is turned off. By pulsing the ultrasound, it is therefore, to controllably release the substance in a pulsed manner corresponding to the ultrasound signal.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.60/134929, filed May 19, 1999, and from International Application underthe Patent Cooperation Treaty PCT/US00/14081, filed May 19, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This work was supported by the NSF VPW grant #9550423, NIH Grant#R0152216, and NIH grant R01 CA76562-01A1.

FIELD OF THE INVENTION

This invention relates to stabilization of micelles, and activation ofmicelles for delivery of substances such as drugs.

BACKGROUND OF THE INVENTION

The efficacy of cancer chemotherapy is limited by toxic side effects ofanticancer drugs. The ideal scenario would be to sequester the drug in apackage that would have minimal interaction with healthy cells, then atthe appropriate time, release the drug from the sequestering containerat the tumor site. To achieve this goal, various long-circulatingcolloid drug delivery systems have been designed during the last threedecades. A common structural motif of all these long circulatingsystems, whether they be nanoparticles, liposomes, or micelles, is thepresence of poly(ethylene oxide) (PEO) at their surfaces. The dynamicPEO chains prevent particle opsonization and render them“unrecognizable” by reticulo-endothelial system (RES). [1] Thisinvaluable advantage has promoted extensive research to develop newtechniques to coat particles with PEO, techniques ranging from physicaladsorption to chemical conjugation.

From the technological perspective, the most attractive drug carriersare polymeric micelles formed by hydrophobic-hydrophilic blockcopolymers, with the hydrophilic blocks including PEO chains. Thesemicelles have a spherical, core-shell structure, with the hydrophobicblock forming the core of the micelle, while the hydrophilic PEO block(or blocks) forms the shell. Block copolymer micelles have promisingproperties as drug carriers in terms of their size and architecture. Theadvantages of polymeric micellar drug delivery systems over other typesof drug carriers include: 1) long circulation time in blood; 2)appropriate size (10 to 30 nm) to escape renal excretion but to allowfor the extravasaation at the tumor site; 3) simplicity in drugincorporation, compared to covalent bonding of the drug to the polymericcarrier and 4) drug delivery independent of drug character. [2]

The ability of PEO-coated particles to prohibit adsorption of proteinsand cells depends on the surface density of PEO chains, their length anddynamics. [1,3] However, only a few known block copolymers form micellesin aqueous solutions. Among them, AB-type block copolymers, e.g.poly(L-aminoacid)-block-poly(ethylene oxide) [2,3-13] and ABA-typetriblock copolymers. Triblock copolymers of this class are availableunder the name PLURONIC™, which shall be referred to generically hereinas “P-triblock”. P-triblocks are block polymers of PEO and PPO, usuallytriblock PEO-PPO-PEO copolymers, where PPO stands for poly(propyleneoxide); the hydrophobic central PPO blocks form micelle cores, whereasthe flanking PEO blocks form the shell, or corona which protectsmicelles from the recognition by RES. P-triblock copolymers arecommercially available from BASF Corp. and ICI. P-triblock polymers arealso disclosed in U.S. Pat. No. 5,516,703 to Caldwell et al, issued May14, 1996, which is hereby incorporated by reference. P-triblockstructure in aqueous solution have been extensively investigated by manyauthors and have been recently reviewed by Alexandridis [22], see also[16]. The phase state of P-triblock micelles can be controlled bychoosing members of the P-triblock family with appropriate molecularweight, PPO/PEO block length ratio, and concentration. The hydrodynamicradii of P-triblock micelles at physiological temperatures range between10 and 20 nm, which makes them prospects as potential drug carriers.

Recently the synthesis of the poly(ethyleneoxide-block-isoprene-block-ethylene oxide) triblock copolymer has beenreported [23]. Isoprene blocks comprising the core of this copolymerwere crosslinked by UV irradiation, rendering micelles stable in thecirculation system of mice.

The incorporation of drugs into block copolymer micelles may be achievedthrough chemical and physical routes. Chemical routes involve covalentcoupling of the drug to the hydrophobic block of the copolymer leadingto micelle-forming, block copolymer-drug conjugates. However, thisapproach involved complex synthetic steps and purification procedures.This concept is disclosed in Rigsdorf, et al. [24] and Kataoka, et al.[7-10, 25-27]

Physical entrapment is a better way of loading drugs into micellarsystems. Physical entrapment of the anti-cancer drug doxorubicin (DOX)in micelles composed of poly(ethylene oxide-block-b-benzyl L-aspartate)has been disclosed by Kataoka, et. al. [12].

Polymeric surfactants at various aggregation state have been tested asdrug carriers. P-triblock molecules in the uniimeric form (below thecritical micelle concentration, CMC) were found to sensitize multi-drugresistant (MDR) cancerous cells. Kabanov and Alakhov [20, 28, 29] havefound that there is a-dramatic increase in Daunorubicin and DOXcytotoxic activity toward the multi-drug resistant cell lines while inthe presence of 0.01 to 1% of PLURONIC P85 or L61. The efficacy of thedrug/P-triblock systems dropped above the CMC. It was concluded that theefficacy of P-triblockdelivery systems was based on the presence ofP-triblock unimers.

The drop in the efficacy of drug/P-triblock systems above the CMC may bedue to the substantial decrease in the intracellular drug uptake fromdense P-triblock micelles. [30-32] The drug incorporated into themicelle core is masked from the external media by the corona composed ofPEO chains.

This phenomenon may be used advantageously to prevent the unwanted druginteractions with healthy cells. However, the challenge is to ensuredrug uptake at the tumor site.

The fundamental difference between using polymeric surfactants below orabove the CMC is that below the CMC the enhanced intracellular uptakeand enhanced cytotoxicity of the drug delivered with P-triblock unimersis exploited [20, 28, 29, 33], whereas above the CMC, the shieldingproperties of P-triblock micelles are used to prevent unwanted druginteractions with healthy cells. To ensure drug uptake from (or togetherwith) polymeric micelles at the tumor site, micelle perturbation andcell membrane permeabilization by ultrasound is being proposed [30-32,34].

Summarizing, drug delivery using micellar drug carriers proved to havemany advantages over the use of free drugs.

Some micellar systems are structurally stable (these are micelles withsolid-like cores that dissociate slowly at levels below their CMC, e.g.micelles formed by poly(L-aminoacid)-block-poly(ethylene oxide)copolymers [2, 5, 26]). As indicated by NMR data, molecular motion inthe core of these micelles is substantially frozen. In contrast,P-triblock micelles or those formed by poly(ethyleneoxide-block-isoprene-block-ethylene oxide) triblock copolymer dissociatevery fast upon dilution [16]. These micelles have “soft” cores, whichmeans that at room temperature theft molecular segments are abovecorresponding glass transition temperature, T_(g) and move relativelyfast. Since upon IV injections, the concentration of the polymeric drugcarrier can drop to levels below the CMC, non-stable micelles requireadditional stabilization to be used in micellar form.

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Objects of the Invention

It is, therefore, an object of the invention to provide a system for thestabilization of micelles.

Another object of the invention is to provide a system for activation ofstabilized micelles.

It is further an object of the invention to provide a system for drugdelivery by the stabilization and activation of micelles.

Further objects of the invention will become evident in the descriptionbelow.

BRIEF SUMMARY OF THE INVENTION

The present invention involves various routes of micelle stabilizationagainst degradation upon dilution. The invention also involves theeffect of ultrasound on drug release from micelles and drug uptake bycancerous cells. In practice of the present invention, a drug can beencapsulated in a long-circulating micelle. Extravasation proceed onlyat tumor sites due higher permeability of blood vessels, and is enhancedby ultrasound. The micelle-encapsulated drug is accumulated at the tumorsite. The uptake of the micelle-encapsulated drug is enhanced byultrasound.

The advantages of the micelle drug carriers of the invention includedlong circulation time in the blood, appropriate size to escape renalexcretion, appropriate size to allow for extravasation at the tumorsite, and simplicity of drug loading. In addition, sterilization ispossible by filtration, and micelles can be introduced by intravenousinjections.

The micelles are formed from any suitable micelle forming blockcopolymer, including AB-type, and ABA-type. Exemplary micelle formingblock polymers are the, polymers of the P-triblock family.

To be used as drug carriers, P-triblock micelles require stabilizationto prevent degradation caused by significant dilution accompanying IVinjection. Three routes of P-triblock micelle stabilization are includedin the present invention The first route is direct radical crosslinkingof micelles cores which results in micelle stabilization.

In the second method, a small concentration of an oil, such vegetableoil (about 0.0005 percent)is introduced into diluted P-triblocksolutions. This substantially decreases micelle degradation upondilution while not compromising drug loading capacity of oil-stabilizedmicelles. The amount oil used is much small than that required to forman emulsion, which is about 1 percent. The oil bonds or interacts withthe core to make it more hydrophobic and stable, but it is insufficientto form an emulsion of the oil in water.

The third route is a technique based on polymerization of thetemperature-responsive LCST hydrogel in the core of P-triblock micelles.The hydrogel phase is in a swollen state at room temperature, whichprovided for a high drug loading capacity of the system. The hydrogelcollapses at physiological temperatures which locked the core ofmicelles thus preventing them from fast degradation upon dilution. Thisnew drug delivery system is referred to herein as “P-gel”. Phasetransitions in P-gel caused by variations in temperature orconcentration were studied by the EPR.

The effect of P-triblock concentration in the incubation medium on theintracellular uptake of two anti-cancer drugs was studied. At lowP-triblock concentrations, when the drugs were located in thehydrophobic environment, drug uptake was increased, presumably due tothe effect of a polymeric surfactant on the permeability of cellmembranes. In contrast, when the drugs were encapsulated in thehydrophobic cores of P-triblock micelles, drug uptake by the cells wassubstantially decreased. This may be used advantageously to preventundesired drug interactions with normal cells. Ultrasonication enhancedintracellular drug uptake from dense P-triblock micelles. These findingspermitted the formulation of a new concept of a localized drug delivery.

An advantage is that the p-gel micelles are stable for drug delivery,but not so stable that they cannot be degraded by the body. After amatter of weeks, the stabilized p-gels will gradually destabilize. Thisallows sufficient time to function effectively as a drug deliverysystem, but the degradation will allow eventual removal from the body.This is unlike many drug delivery systems that involve stable componentsthat are slow to be removed from the body. The thermodynamics of thep-gel system direct the system toward dissolution, and instability, butthe kinetics are very slow.

Another aspect of this embodiment is the use of hydrogels that arestimuli responsive to other environmental states, such as pH.

Other substances that are introduced into the body, other than drugs,can be encapsulated and delivered by the stabilized micelle system ofthe invention.

Ultrasound Release

Another aspect of the invention is the use of pulsed ultrasound torelease an encapsulated drug. In particular, for hydrogel stabilizedmicelles, the release of drug by ultrasound is reversible, with allows ahighly controlled release of drug using a pulsed ultrasound system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a, 1 b, 1 c. 1. 3-dimensional size exclusion chromatograms ofP-triblock samples: x-axis—retention time, mm; y axis—wave length, nm;z-axis—absorption. Concentration of the injected polymer 1 wt %;eluent—water, detector: UV diode array; injection volume 10 μl;temperature 30° C. (a) control P-triblock sample, (b) and (c)crosslinked samples using 5 wt % (b) and 10 wt % (c) of benzoyl peroxidein P-triblock Micelles.

FIG. 2. The concentration of a solubilized 16-DS vs. P-triblock PLURONICP405 concentration in aqueous solutions. The EPR spectra are shown for16-DS in a 0.01 wt % (lower spectrum) and 10 wt % P-triblock solution(upper spectrum) at room temperature.

FIG. 3. The EPR spectra at room temperature of 16-DS in controlP-triblock solutions of progressively decreasing concentrations.

FIG. 4. The EPR spectra at 37° C. of 16-DS dissolved in oil (spectrumA), solubilizedin 1% oil-in-water emulsion stabilized by 0.01%P-triblock PLURONIC P-105 (spectrum B), and solubilized in 1%oil-in-water emulsion stabilized by mucin (spectrum C).

FIG. 5. EPR spectra at room temperature (left) and 37° C. (right) of16-DS in a 0.1% Plumnic solution (left) or 1% P-triblock solution(right) in the absence (up) or presence (down) of oil; oilconcentrations are indicated next to the respective spectrum. Thespectra indicate that in the presence of oil in diluted P-triblocksolutions, the predominant fraction of the probe (more than 90% for a0.1% solution) is localized in the hydrophobic environment of P-triblockmicelle cores characterized by a˜=14.60.

FIG. 6. A schematic diagram showing the interpenetrating network of PPOblocks and hydrogel molecules.

FIG. 7. A schematic diagram showing the reversible collapse andreswelling of a hydrogel stabilized micelle.

FIG. 8. The EPR spectra of 16-DS solubilized in 10% P-105/1%poly(NiPAAm) nanoparticles. At room temperature (upper spectrum), theprobe is localized predominantly in the hydrophilic environment of aswollen hydrogel; heating the P-gel solution to 37° C. (above hydrogel'sLCST) results in probe re-distribution: 94% of the probe is transferredinto the hydrophobic environment (lower spectrum).

FIG. 9. The EPR spectra of 16-DS in a 10-fold diluted initial P-gelsolution (final concentration 1% P-triblock/0.1% poly(N1PAAm). In adiluted solution, the hydrogel phase has much lower microviscosity thanin the initial P-gel solution (compare upper spectrum of this Figure tothat of FIG. 8). Spectral changes with increasing temperature indicateprogressive micellization of a diluted P-gel solution.

FIG. 10. The EPR spectrum at room temperature of Rb (0.1 mM) solubilizedin 10% P-triblock solution showing the superposition of signals arisingfrom two drug populations differing in motion intensity. Upperspectrum—experiment; middle—simulation using the ENVoigt program;lower—differential spectrum between the experimental and simulatedspectra. Both signals correspond to the probe located in thehydrophilic-environment; sharp spectrum corresponds to a probe withfaster motion.

FIG. 11. W-band EPR spectrum at room temperature of a 100-fold dilutedinitial P-gel solution manifesting probe transition from the hydrophilicto the hydrophobic environment upon dilution, upper spectrum—experiment;middle—simulation; lower spectrum—differential spectrum between theexperimental and simulated spectrum (a sharp right line is a Ms^(2˜)standard).

FIG. 12. Is a chromatogram showing the molecular weight of a NNDEAP-gel.

FIG. 13 Is graph showing preservation of hydrophobic cores of micellesat very high dilutions of P-gel samples.

FIG. 14. A scheme showing conjugation of nitroxide radical(1-oxo-2,2,6,6-piperidone-4-hydrazone) to DOX molecule to form Rb.

FIG. 15 . A graph showing polymerized NNDEA light scattering.

FIG. 16. A graph showing particle sizes of NNDEA stabilized micelles at37° C.

FIG. 17. Effect of sonication on the intracellular uptake of DOX byHL-60 cells: fluorescence of HL-60 cell lysates, normalized to the cellconcentration, as a function of P-triblock PLURONIC P-105 concentration;([DOX]=3.4 μg/ml, incubation/sonication time 1 h).

FIG. 18. Experimental arrangement for fiberoptic detection offluorescence of drug under ultrasound exposure. For 20 kHz exposure, thetransducer was controlled by different electronics and was inserted intothe exposure bath from above.

FIG. 19. Example of release profiles of DOX from a 10% P-triblocksolution and from PBS. Raw and Fourier-filtered data are presented forthe 10% P-triblock solution. For the PBS solution, ultrasound was turnedon at 60s and off at 120s; there was a negligible change of DOXfluorescence under sonication.

FIG. 20. Rb release profile from 10% P-triblock P-105 micelles at CW andpulsed sonication; Rb concentration 20 μg/ml; ultrasound frequency 47kHz, power. density 3.5 W/cm².

FIG. 21. DOX release profiles from 10% P-triblock P-105 micelles at CW(a) and pulsed (b-e) sonication; DOX concentration 6.7 μg/ml; ultrasoundfrequency 20 kHz, power density 0.058 W/cm²; pulse sequence: (b)-0.1 s“on”: 0.1 s “off”; (c)-0.5 s:0.5 s; (d)-1 s:1 s; e-1 s:3 s.

FIG. 22. DOX release from 0. 1% and 10% P-triblock micelles at 20 kHz;DOX concentration 6.7 μg/ml.

FIG. 23. Effect of temperature on Rb. fluorescence intensity in PBS andP-triblock PLURONIC P-105 solutions. [Rb]=10 μg/ml.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES Manufacture of StabilizedMicelles

P-triblock PLURONIC P-105 micelles were chosen as a model polymericmicellar system. The rational behind the choice of P-triblock was thatP-triblock can form unimers, loose water penetrated aggregates, andmicelles with hydrophobic cores, and the phase state of the drug carriercan be controlled by choosing members of the P-triblock family withappropriate molecular weight, PPO/PEO block length ratio, andconcentration. The phase state of P-triblock solutions can becharacterized by the Electron Paramagnetic Resonance technique (EPR)[16]. In addition, reports have indicated that P-triblock solutions inconcentration below 1 wt % are non-toxic [14].

The hydrodynamic radii of P-triblock micelles at physiologicaltemperatures range between 10 and 30 nm, which makes them prospectivedrug carriers.

Experimental Methods Materials

P-triblock P-105 with an average molecular weight of 6,500, the numberof monomer units in PEO and PPO segments being respectively 37 and 56,was supplied by BASF Corporation and used as received. Spin probe16-doxylstearic acid (16-DS) was purchased from Sigma, St. Louis, Mo.Monomers, crosslinkers, radical initiators were purchased fromPolysciences, Inc., Warrington, Pa.

Cells

HL-60 promyelocytic cell line was provided by Dr. Murray (Brigham YoungUniversity, Provo, Utah), They were cultured in RPMI 1640 mediumsupplemented with 20% fetal calf serum, 2 mM L-glutamine, 0.2% sodiumbicarbonate and 50 μg/ml gentamicin at 37° C. in humidified aircontaining 5% CO₂.

Drugs

Ruboxyl (Rb) was provided by Dr. Shapiro (Institute of BiochemicalPhysics, Moscow, Russia). DOX was supplied by the University Hospital,University of Utah, Salt Lake City, Utah, USA).

Incubation Media

Cells were incubated with the drug in the RPMI medium or PBS in theabsence or presence of P-triblock P 105. P-triblock was dissolved at0.1, 1.0, 10, or 20 wt % in RPMI media or PBS and the solutions obtainedwere sterilized by filtration through a 0.2 um filter.

Size Exclusion Chromatography

Size exclusion chromatography was performed on a Spherogel TSK 600OPW(Beckman Co), with water as a mobile phase (flow rate of 1 ml/mm at 30°C.); injection volume was 10 μl. Hewlett Packard Series 1100 LiquidChromatograph equipped with multi-wavelength detector was used, whichallowed to measure absorption of analytes in the range of 190-400 nm.The column was calibrated using PEG standards. Before the experiment,control and crosslinked P-triblock samples were diluted to the finalP-triblock concentration of 1% w/v.

Measuring Loading Capacity of Micelles

A dry powder of the EPR probe (16-DS) was incubated with unstabilized orstabilized micellar solutions of P-triblock for 15 mm under constantshaking. Unsolubilized probe was removed by centrifugation. Theconcentration of a solubilized probe was measured by the EPR. The sametechnique of probe introduction was used for pyrene. Water solubility of16-DS or pyrene is very low and can be neglected. Dissolved probes areassociated with P-triblock molecules and thus reflect the loadingcapacity, dynamics and environment of the latter. For a fluorescentdrug, Rb, the same concentration of Rb (20 μg/ml) was introduced intonon-stabilized and stabilized micellar P-triblock solutions at 37° C.from a stock solution. Because fluorescence of Rb within P-triblockmicelles is much higher than outside micelles [31], the fraction of thedrug within micelles can be estimated as

F _(mes) =a _(m) F _(m)+(1−a _(m))F _(s)

where Fmes is a measured fluorescence intensity, F_(m) is Rbfluorescence intensity when all the drug is localized in the hydrophobiccore of micelles (measured in a 20% P-triblock solution at 37° C.),F_(s) is fluorescence intensity in non-micellar solutions (i.e., inPBS), and a_(m) is the fraction of the drug located in the hydrophobiccore of micelles.

Spin Probe Introduction

A 16-DS spin probe was introduced into P-triblock solutions in thefollowing manner: an aliquot of a stock solution of 1 6-DS in ethylalcohol was placed at the bottom of a test tube; alcohol was evaporatedin the air stream, upon which P-triblock solutions of variousconcentrations were added. The samples were sonicated for 15 mm toenhance spin probe solubilization.

EPR Experiments

The samples were placed in the EPR capillaries or flat EPR cells (WilmadGlass Corporation, Buena, N.J.). EPR spectra were acquired in a Bruker(Billerica, Mass.) ER-200 SRC X-band EPR spectrometer (installed at theUniversity of Utah) or Varian (Palo Alto, Calif.) Century Series E-112X-band spectrometer in the

Illinois EPR Research Center (University of Illinois atUrbana-Champaign). Incident microwave power was set to 0.5-2 mW to avoidsaturation. Modulation frequency was 100 kHz, modulation amplitude was aquarter of a line width. EPR spectra were recorded at room temperatureand at 37° C. The EPR spectra were taken sequentially, digitized, andstored with the aid of a commercial EPR software/hardware package(Scientific Software Services, Bloomington, Ill.).

Double integrals of individual EPR signals are proportional to thecorresponding spin concentrations. Hyperfine splitting of spectral linescharacterizes the hydrophobicity of a probe environment; signal shape(pick-to-pick line width, low-field to high-field line intensity ratio)is used to measure probe motion parameters (rotational correlation time,t_(r), anisotropy of motion etc.). Lorenzian component of the line shapeand double integrals of the spectra were measured by fitting of eachspectrum to the inhomogeneous line shape model using a computer programdescribed in [35]. This program provides for the separation ofoverlapping EPR signals. All spectra were processed in automatic mode,in which the best-fit parameters for the spectrum were used as aninitial approximation for the Levenberg—Marquard optimization of thenext spectrum in the sequence.

Dynamic Light Scattering

Dynamic light scattering was measured using a BI 200 Spectrometer fromBrookhaven Instruments equipped with a BI 2030 AT 72-channelautocorrelator. DSC data were analyzed using a BI30AT program.

Insonation

Ultrasound was generated by a Sonicor SC 100 sonication bath operatingat 67 kHz and 37° C. Power density was controlled by adjusting the inputvoltage and was measured with a hydrophone.

Measuring Drug Uptake by the Cells

Intracellular uptake of DOX and Rb was measured using a fluorescencetechnique, in which compounds were excited at 488 nm and technicalemission spectra were recorded between 510-700 nm. Two sets of sampleswere studied, one incubated and another sonicated. For the first set(incubated), the cells were incubated at 37° C. with DOX or Rb, whichwere either dissolved in the RPMI medium (or PBS), or the drugs weresolubilized in P-triblock PLURONIC P-105 solutions of variousconcentrations. For the second set of samples, the cells were sonicatedby 70 kHz ultrasound at 379C up to 1 hour in the presence of drug toassess the, effect of ultrasound on the drug uptake from molecular andmicellar solutions. After being incubated/sonicated with and without thedrug, the cells were spun out, washed twice with cold PBS, andre-suspended in PBS. Sonication power density was maintained at or below2.4 W/cm². No immediate cell death caused by sonication was observed.Sonication in the absence of P-triblock did not, affect cellproliferation. Because drug fluorescence within the cells wassubstantially quenched, drug uptake was quantified in cell lysates;cells were lysed by incubating them with 1 wt % SDS solution for 1-2 hat37° C. This process transferred the drug from cellular, components toSDS micelles. Calibration experiments showed a linear dependence of Rbor DOX fluorescence intensity on concentration in 1% SDS solutions inthe concentration range of interest. Upon the completion of cell lysis,fluorescence spectra of the lysates were recorded. To quantify theconcentration of lysed cells, cell lysates were filtered through 0.2 mmfilters, and their optical density was measured by protein absorption at280 nm (OD 280 nm). Calibration experiments showed a linear dependenceof OD 280 nm on the concentration of lysed cells. The fluorescenceintensity of lysates was normalized by OD 280 nm. In parallel, thedepletion of the drug from the incubation medium was measured by thedecrease of supernatants' fluorescence.

Results and Discussion Micelle Stabilization Against Degradation UponDilution Direct Radical Crosslinking

At low temperatures and/or concentrations, P-triblock exists in aqueoussolutions as individual coils, or unimers, with a size of approximately1-2 nm [36]. Upon increasing temperature and/or concentration, thetransition proceeds from unimers to loose, water penetrated aggregatesto micelles with hydrophobic cores. Micelle cores consist of PPO blocks.Upon dilution below the CMC, these micelles dissociate into loose,multimolecular aggregates or unimers within minutes. (See Rolland [23].)

This experiment shows that radical crosslinking of P-triblock micellecores will prevent micelle dissociation. The crosslinking procedure isdesigned to confine the crosslinking to micelle cores withoutcompromising the structure and dynamics of the PEO side chains. Forthat, a hydrophobic radical initiator was chosen, benzoyl peroxide, thatdissolves predominately in the micelle core. The initiator at theconcentration range of 0.5 to 20 mg/ml (which corresponds to 0.25 wt%-10 wt % in respect to P-triblock) Was introduced, into 20% P-triblockPLURONIC P-105 solution under sonication (30 sec, 70 kHz). The solutionwas degassed and the crosslinking was performed by heating at 60° C. for24 hours. Micelle stabilization upon dilution was tested by sizeexclusion chromatography.

The chromatograms of non-crosslinked samples manifested two major peaksat retention times of 7.6 and 11.4 mm and a very weak-peak at 9.2 mm(FIG. 1a and Table 1). UV-spectra of all spectra components were in thewavelength area of polyethers' absorption (190-210 nm). UV spectrum ofthe first peak had a long tail extending to 400 nm. It is speculatedthat this peak belongs to swollen multimolecular aggregates and that along tail in UV spectra of these components results from lightscattering.

For the third peak, no tails beyond 210 nm were observed. This peakpresumably belongs to unimers and characterizes the molecular weightdistribution of the initial polymer that is found rather broad(corresponding to molecular weights from 600 D to 23,000 D, with amaximum at 7,000 D, which is close to .6,500 D reported for P-triblockPLURONIC P105 by the manufacturer).

The chromatograms of crosslinked samples manifested all three peakscharacteristic of the control non-crosslinked sample. However, the ratioof peak area changed dramatically upon crosslinking, a peak at aretention time of about 9.0-9.3 mm becoming the strongest (FIG. 1b). Thearea of this peak substantially increased (to about 80% of total) uponincreasing initiator concentration, while areas of the first and thethird peak dropped. UV spectra manifested a presence of benzoyl peroxidein the component corresponding to the strong second peak (absorption at226 nm, FIGS. 1b and 1 c). This implies that the second peak belongs toP-triblock micelles stabilized by radical crosslinking. Theconcentration of crosslinked micelles grew with the concentration ofbenzoyl peroxide (Table 1).

TABLE 1 Parameters of (PC peaks for non-crosslinked and crosslinkedP-triblock P-105 samples. Sample Relative peak area (%‘/Retention time(min’ Control 56/7.6  5/9.2 39/11.4 Crosslinked˜1* 30/7.7 44/9.0 26/11.3Crosslinked˜2*  9/7.5 78/9.3 13/11.4 *The concentration of benzoylperoxide was 5 wt % of P-triblock for a crosslinked-1 sample and 10 wt %of P-triblock for a crosslinked-2 sample.

Introduction of Low Concentrations of Vegetable Oil

The rationale behind this approach was that increasing hydrophobicinteractions in the micelle core should result in decrease in the CMCand thus prevent micelle degradation upon dilution. In these experimentsthe EPR technique was used to characterize micelle formation anddegradation. This technique has been successfully used to study themicellization of various members of P-triblock copolymer family inaqueous solutions [16] and dynamics of P-triblock-coated polymericcolloids [3].

The spin-probe EPR technique provides the following information on thesystem: solubilization efficiency (the concentration of a solubilizedprobe measured by the double integral of the spectral line), polarity ofthe probe environment (characterized by the hyperfine splittingconstant, a_(N)), the microviscosity of the probe environment(characterized by the rotational correlation time, t_(rot)), and localconcentrations of the probe (characterized by the line width or a shapeof the spectrum at 77K).

Formation of P-triblock micelles resulted in dramatic changes of EPRspectra of a solubilized spin probe, 16-DS (FIG. 2). This probe has anamphiphilic character; two polar groups, namely carboxyl and nitroxidegroups, are separated by a long fatty chain. The probe does notspontaneously dissolve in water; dissolved probe molecules areassociated with P-triblock molecules and thus reflect the loadingcapacity, dynamics and environment of the latter. Probe “solubility” inmicellar solutions is much higher than that in unimeric solutions (seeFIG. 2), which allows the probe to accurately report the onset ofmicelle formation.

EPR spectra of solubilized nitroxide probes are presented by three-linesignals; line shape (hyperfine splitting constant a_(N), peak-to-peakamplitude ratio, line width) reports hydrophobicity and microviscosityof the probe environment. Differences in the EPR spectra of a 16-DSprobe solubilized in P-triblock P-105 solutions of variousconcentrations indicated probe transition from the hydrophilicenvironment of P-triblock unimers (FIG. 3, lower spectrum) to thehydrophobic environment of cores of P-triblock micelles (FIG. 3, upperspectrum; see also FIG. 2). At a very low P-triblock PLURONIC P-105concentration corresponding to P-triblock unimers (0.001 wt %), oneP-triblock molecule was associated with several 16-DS molecules, whichwere located in the hydrophilic environment. Upon increasing P-triblockconcentration up to 0.1% at room temperature, several P-triblockmolecules corresponded to the solubilization of one probe molecule; theprobe was still located in the hydrophilic environment; which system iscalled “loose aggregates”. Finally, at P-triblock concentrations of 1 wt% or higher, EPR spectra showed probe transfer into the hydrophobicenvironment indicating the formation of P-triblock micelles withhydrophobic cores [16]. The EPR spectra presented in FIG. 2 and FIG. 3indicated that the microviscosity of the interior of P-triblock micelleswas substantially higher than that outside micelle cores; they alsoshowed a dramatic increase of P-triblock solubilization efficiency forlipophilic substances upon the onset of the formation of micelles withhydrophobic cores.

For the oil-stabilized micelles, oil (1% v/v) was introduced into a 20%(w/v) P-triblock solution together with a spin probe, 16-DS; the samplewas sonicated for 15 mm, upon which sequential dilutions were done tothe final P-triblock concentrations of 10%, 2%, 1%, 0.1% and 0.01%. Inparallel, sequential dilutions were made to the control sample that didnot comprise oil.

The EPR spectra of 16-DS dissolved in oil (spectrum A) or solubilized inoil-in-water emulsions stabilized with a surface active protein, mucin(B) are presented in FIG. 4.

The shape of the spectrum of 16-DS solubilized in a 10% P-triblockPLURONIC P-105 solution (FIG. 3, upper spectrum)as well as that of aprobe dissolved in oil (FIG. 4, upper spectrum) is characteristic of anearly isotropic probe motion in a viscous hydrophobic medium. Values ofhyperfine splitting constants a_(N) indicate that oil is morehydrophobic than a core of P-triblock micelles (a_(N)=14.270 for 16-DSin oil to be compared to 14.60 for a probe in a 10%P-triblock-solution).

For control samples, the shape of spectral lines changed dramaticallyupon progressive dilutions indicating micelle degradation.

In 10% P-triblock solution, at room temperature, the probe was localizedexclusively in the hydrophobic core of P-triblock micelles (a_(N)=14.60). Upon a 5-fold dilution of this sample, a very small populations ofthe probe located in the hydrophilic environment was revealed (about 1%of the total probe), indicating the onset of micelle degradation.(Spectra simulations-were done using the EWVoigt software [Peef(PhilipD., II) Morse, Scientific Software Services, P.O. Box 406, Normal, Ill.61761-0406].) This conclusion is based on the following consideration:since the probe is always associated with P-triblock molecules, theconcentration ratio of the probe in the hydrophobic and hydrophilicenvironment reflects the ratio of micelle-encapsulated andunimer-associated (or loose aggregates-associated) probe. In dilutedsolutions, EPR spectra manifested the superposition of the linescorresponding to the probe localized in the hydrophobic environment ofmicelle cores (broad lines with a_(N)=14.60) and in the hydrophilicenvironment of unimers or loose aggregates (sharp lines with a_(N)=15.90). The population of the probe in the hydrophilic environment grew withdilution. Only traces of micelles were observed in 0.1% solutions (abroad single line that appears as a background of a three-line signalfor 0.1% P-triblock concentration is characteristic of a very high localconcentration of the probe in a small number of micelles). No micellesmanifested themselves in 0.01% solutions, which means that thisconcentration was below the CMC; the probe was located exclusively inthe hydrophilic environment characterized by a_(N)=16.00; a sharpthree-line spectrum pointed to fast tumbling.

When 1% v/v oil was introduced into a 0.01% P-triblock solution, atypical oil-in-water emulsion was formed (compare the second and thethird spectrum of FIG. 4) indicating that at this P-triblock-to-oilratio, P-triblock stabilized oil-in-water emulsions.

In contrast, when oil-to-P-triblock ratio was low (0.005-0.05% v/v oilin 0.1%-1% P-triblock solutions), oil did not form emulsions but wasdissolved in the core of P-triblock micelles, which resulted in micellestabilization (FIG. 5). In the presence of low concentration of oil, atroom temperature, even in 0.01% or 0.1% P-triblock solutions, thepredominant fraction of the probe was localized in the hydrophobicenvironment of P-triblock micelle cores characterized by a_(N)=14.6 G,while in the absence of oil only a negligible concentration of the probein the hydrophobic environment was found.

The most interesting information provided by the EPR spectra of 16-DSsolubilized in diluted P-triblock solutions containing a lowconcentration of oil is that the structure of these solutions was thatof P-triblock micelles rather than oil microemulsions (compare B and Dspectra of FIG. 5 with B and C spectra of FIG. 4).

In conclusion, the introduction of low concentrations of vegetable oilstabilized micelles against degradation upon dilution. Introduction ofoil did not compromise solubilization efficiency of P-triblock micellesfor anticancer drugs.

Micelle Stabilization by Developing the Interpenetrating Network of aPolymeric Surfactant and a Temperature-responsive LCST Hydrogel

This experiment shows a novel synthetic pathway to stabilize P-triblockmicelles by polymerizing a temperature-responsive low critical solutiontemperature (LCST) hydrogel in the micelle core. The hydrogel-formingpolymer produced the interpenetrating network inside the core ofP-triblock micelles. (See FIG. 6) The rational behind this approach wasthat at room temperature the LCST hydrogel was in a swollen state, whichprovided for a very high drug loading capacity for lipophilic andhydrophilic drugs. At physiological temperatures, themicelle-encapsulated gel collapsed, “locking” the core of the micellethus preventing micelles from rapid degradation upon dilution. (See FIG.7) This new drug delivery system is known under the trademark PLUROGEL,which will be generically referred to herein as P-gel. P-gel is asterically protected nano-dispersed hydrogel.

The sizes of P-gel particles were measured by dynamic light scattering.P-gel particles were larger than P-triblock micelles (particle diameterranged between 30 nm and 400 nm depending on P-triblock concentration,temperature, and type of a gel-forming monomer. This is to be comparedto 12-15 nm for P-triblock micelles at 37° C.). An example of particlesizes for P-gel particles is shown in FIG. 16. P-gel particle size isadvantageous for drug delivery applications.

An example of a protocol of P-gel polymerization is given below.N-isopropylacrylamide (NiPAAm) (NI was carried out in inert atmosphereat 70° C. for 24 hours. Upon termination of polymerization, theunreacted monomer, crosslinking agent, and initiator was separated bydialysis through a 1000 D cutoff membrane against PBS.

Obtained P-gel solutions were clear at room temperature. They manifesteda sharp, completely reversible transition at 32° C. (e.g. at LCST for ahydrogel), characterized by a formation of a very stable slightly milkydispersion.

When a monomer was polymerized without P-triblock, it precipitated froma reaction medium at any temperature.

When polymerized without neither P-triblock nor a crosslinker, thepolymer precipitated from a reaction medium, but could be solubilized byaddition of P-triblock, with a formation of a milky dispersion.

In another example, N,N-Diethylacrylamide (NNDEA) was polymerized in thepresence of P-triblock P-105 micelles. The polymerizations resulted inan interpenetrating network of NNDEA and P-105 that stabilizes themicelles at concentrations below the critical micellar concentration offree P-105. The NNDEA was crosslinked with N,N′-Bis(acryoyl)cystamine(BAC) and the degree of micellar stability was determined using dynamiclight scattering and the fluorescent probe diphenylhexatriene (DPH). Theincreased micellar stability was not permanent and disappeared over atime period of days to weeks.

40 ml of double distilled water containing 10 wt % P-triblock P-105 wasadded to a round bottom flask. N,N-diethylacrylamide (NNDEA) monomer wasadded to give concentrations ranging from 0 to 1 wt % monomer. BAC wasadded as a crosslinking agent to give BAC:NNDEA mole ratios ranging fromzero to 1:20. AIBN was added as an initiator and the flask was connectedto a water condenser and purged with nitrogen for at least one hour. Thesystem was then allowed to polymerize for 24 hours at 65° C. withmagnetic stirring and a continuous nitrogen purge.

Molecular Weight Determination

The molecular weight of un-crosslinked p(NNDEA) was investigated by gelpermeation chromatography using a Waters GPC system (Milford, Mass.)(model 515 pump with styragel columns and a model 2410 refractive indexdetector). Polymerization samples were dried, dissolved intetrahydrofuran, and filtered through a 0.22 μm teflon filter beforebeing injected into the GPC system. Molecular weights were determinedusing polystyrene standards and Water's Millennium³² software.

Molecular weight for NNDEA P-gel was found to be 21,974. (See FIG. 12).

NNDEA P-Gel Particle Stabilization and Micro-environment

The critical micellar concentration (CMC) and micellar stability werestudied for NNDEA p-gel using a fluorometer with DPH as a fluorescentprobe. The emission spectra of DPH is highly dependent upon thehydrophobicity of the local environment and is therefore useful fordetermining the presence of a hydrophobic environment. Samples wereserially diluted in double distilled water to give P-triblock P-105concentrations ranging from 10 wt % to 0.0001 wt %. DPH was added togive a final DPH concentration of 0.1 μg/ml. The samples were excited at360 nm and the emission at 430 nm was measured. The temperature wascontrolled with a re-circulating thermostatic bath connected to thecuvette holder. The results show the noticeable preservation ofhydrophobic cores at very high dilutions of P-gel samples. (FIG. 13)

Turbidity Measurements

The collapse of p(NNDEA) into a hydrophobic state at its LCST is visibleas an increase in the turbidity of an aqueous solution as the resultingparticulates scatter visible light. P-triblock P-105 micelles havediameters much smaller than the wavelength of visible light; andtherefore do not increase the turbidity of the solution. The absorbanceof 600 nm light was used as a means of determining whether or not thep(NNDEA) had aggregated into particles large enough to scatter light.FIG. 15 shows that as NNDEA is polymerized in the presence of increasingconcentrations of P-triblock P-105 the increase in turbidity as thetemperature is raised past the LCST begins to disappear. At aconcentration of 10 wt % P-triblock P-105 and 1 wt % NNDEA there isalmost no increase in the turbidity upon heating. It is believed that atthese concentrations most of the NNDEA has polymerized totally withinthe micellar cores and does not aggregate with other particles uponthermally induced collapse.

Phase State of P-gel Nanoparticles

To characterize phase state of P-gel nanoparticles at varioustemperatures, the EPR and fluorescence techniques were used with 16-DSas a spin probe and DPH as a fluorescent probe, respectively. The probewas solubilized in the initial P-gel solution (10% P-triblock/1%poly(NiPAAm)) or 10% P-triblock/1% poly (NNDEA) at room temperature.

The results of this study are summarized below.

NiPAAm P-gel

Effect of temperature. The presence of a swollen poly(NiPAAm) gel in a10% P-triblock solution hampered the formation of P-triblock micelles atroom temperature. For a 10% P-triblock solution without a gel, the probewas localized exclusively in the hydrophobic environment of P-triblockmicelles characterized by a hyperfine splitting constant a_(N)=14.6 Gand a rotational correlation time, t_(rot)=1.165 ns (see Table 2 andFIG. 3, upper spectrum). In contrast, in a 10% P-triblock/1%poly(NiPAAm) P-gel solution, the probe partitioned hydrophobic andhydrophilic microphases, with a predominant (more than 90%) localizationin the hydrophilic environment (a_(N)=15.5 G) (FIG. 8, upper spectrum).Probe motion in the hydrophilic phase of a P-gel was much morerestricted than in the aqueous phase outside P-triblock micelles(t_(rot)=0.49 ns in a P-gel vs. trot<0.1 ns in a hydrophilic phase ofdiluted P-triblock solutions). This implies that at room temperature, ina P-gel, the probe was located predominantly in the swollen hydrogelmicrophase.

Heating a 10% P-triblock/1% poly(NiPAAm) P-gel solution to 37° C. (i.e.above the LCST of the hydrogel, which is 32° C.) resulted in probere-distribution. The predominant fraction of the probe (94%/o) waslocated in the hydrophobic environment (a_(N)=14.6 0, t_(rot)=0.59 ns)(FIG. 8, lower spectrum). The EPR parameters of this phase were equal tothose for P-triblock micelles (Table 2). About 6% of the probe remainedin the hydrophilic phase, which was characterized by lowermicroviscosity than at room temperature.

The hydrophobic environment of the probe was presumably that of the coreof P-gel nanoparticles that comprised P-triblock micellesinterpenetrated by the network of a collapsed hydrogel.

Above the LCST of the hydrogel, P-gel formed stable dispersions that didnot precipitate; this implies that P-gel nanoparticles were stabilizedby PEO chains on their surfaces.

It appears important for the drug delivery applications that a smallfraction of the probe (6%) was expelled from the core of the particleswhen the gel collapsed. It implies that for a lipophilic drug, a smallfraction of the drug could be released into the environment upon thecollapse of the gel; this fraction of the drug will be taken up by thecells via regular mechanisms typical of a particular drug/cell system.This may provide for a precise external control of drug delivery. Forinstance, the transition temperature of a gel may be set slightly abovethe physiological temperature; the repeated heating/cooling cycles ofthe tumor volume (e.g. by pulsed ultrasound) would result in acontrolled drug release from nanoparticles.

Effect of a P-gel dilution. At room temperature, dilution of the initial10% P-triblock/1% poly(NiPAAm) P-gel solution resulted in a drop ofmicroviscosity of the hydrophilic phase; a rotational correlation timefor the probe in the hydrophilic phase decreased from 0.48 ns to 0.10 nsupon a 10-fold dilution, For a 100-fold diluted P-gel solution, probemotion in the hydrophilic phase was even faster (t_(rot)=0.08 ns)indicating progressive hydration of a gel phase. No hydrophobic phasewas observed in diluted P-gel solutions at room temperature.

Upon heating above the LCST of the hydrogel, a hydrophobic phaseappeared in diluted P-gel solutions, and a temperature-dependentequilibrium was established between hydrophobic and hydrophilicmicrophases. This is clearly manifested in FIG. 9 for a 1% P-105/0.1%poly(NiPAAm) P-gel solution. The fraction of the probe in thehydrophobic phase increased dramatically with increasing temperatureindicating progressive formation of the particles with hydrophobiccores. When compared to control P-triblock solutions of equalconcentrations (without a gel), the fraction of the probe in thehydrophobic phase was always higher in a P-gel than in P-triblock.According to the results of spectra simulation, at 37° C., in a 100-folddiluted initial P-gel solution (final concentration 0.1%P-triblock/0.01% poly(NiPAAm), 86% of the probe was still retained inthe hydrophobic phase of the P-gel, whereas in non-stabilized P-triblocksolutions of the same concentration, the probe was localizedpredominantly in the hydrophilic environment. Even upon 1000-folddilution of the initial P-gel solution, about 33% of the probe remainedin the hydrophobic environment, indicating the preservation ofhydrophobic cores of nanoparticles; no micelles were preserved innon-stabilized P-triblock solutions of equal concentration (Table 2,).

TABLE 2 Rotational correlation time t_(corr), hyperfine splittingconstant a_(N) and a fraction of a probe in the hydrophobic environmentfor a 16-DS spin probe. t_(corr)(ns)/a_(N)(G) t_(corr)(ns)/a_(N)(G) %Sample T° C. hydrophobic hydrophobic hydrophobic P-105 10% RT 1.17/14.6— 100%  P-gel 10%* RT — 0.48/15.5 <10%  P-gel 1% RT — 0.10/15.9   0%P-gel 0.1% RT — 0.08/15.9  0% P-105 10% 37° C. 0.59/14.6 — 100%  P-105,0.1% 37° C. 0.60/14.7 — 54% P-105 0.01% 37° C. — <0.1/15.6  0% P-gel 10%37° C. 0.59/14.6 — 94% P-gel 1% 37° C. — — 99% P-gel 0.1% 37° C.0.80/14.5 — 86% P-gel 0.01% 37° C. — — 33% *Percentage indicatesP-triblock weight concentration. A concentration of a gel-formingpolymer is 10-fold lower.

It is noteworthy that for the same P-triblock concentration, theproperties of the hydrophobic phase in a P-gel differed from those inP-triblock: the environment of the probe was more hydrophobic and themotion of the probe was more restricted in a P-gel (see Table 2.).

These data indicate that the collapse of the hydrogel upon heating aP-gel above gel's LCST results in tighter packing of molecules and lowerdegree of hydration of the cores of P-gel nanoparticles when compared tothose in P-triblock micelles.

In conclusion, developing the interpenetrating network of the LCSThydrogel in the core of P-triblock micelles stabilizes hydrophobic coresof P-gel nanoparticles. Drug loading experiments showed that drugloading capacity of P-gel nanoparticles was higher than that ofP-triblock micelles.

P-gel particles are expected to have a long circulation time in bloodsince they have protective poly(ethylene oxide) chains on their surface.

The properties of P-gel nanoparticles described above make themexcellent prospects as carriers of lipophilic drugs.

Drug Distribution and Release from P-triblock Micelles

Two DNA intercalating anti-cancer drugs, DOX and Rb were used in thisstudy. DOX is widely used in clinical practice as a chemotherapeuticagent. However, like other anti-cancer drugs of anthracyclin family, DOXis cardiotoxic due to the induced production of active oxygen radicals[38, 39]. To reduce a cardiotoxicity, a paramagnetic Tempo-typenitroxide radical (1-oxo-2,2,6,6-piperidone-4-hydrazone) was conjugatedto DOX molecule to form Rb (FIG. 14) [40]. The nitroxide moiety inposition 14 served as a radical trap. A unique Rb molecule is bothfluorescent and paramagnetic, which allows fluorescence and EPRspectroscopy to be used independently in investigations of drug uptake,distribution and metabolism. This makes Rb a powerful research tool.

Rb was used as a spin- and fluorescent probe to monitor drugdistribution in P-triblock micelles and P-gel nanoparticles.

Drug Localization in P-triblock Micelles in the Absence of a Hydrogel

At room temperature, in 10% P-triblock PLURONIC P-105 solutions, EPRspectra revealed two populations of Rb molecules characterized byn_(A)=16.3 G and n_(A)=16.1 G respectively, with a molar ratio of 1:2.5(FIG. 10). EPR signals produced by both Rb populations corresponded tomolecules located in the hydrophilic environment, one being slightlymore hydrophilic than the other (for Rb in PBS, the hyperfine splittingconstant n_(A)=16.4 G). The population of the probe in a morehydrophilic environment (n_(A)=16.3 G) was highly mobile (t_(rot)=0.11ns) and was presumably localized close to the corona/aqueous interfaceof P-triblock micelles. The second drug population (n_(A)=16.1 G) wascharacterized by a much more restricted dynamics (t_(rot)=1.35 ns) andwas presumably localized at the interface between the core and corona ofP-triblock micelles. This conclusion follows from comparison of theresults of EPR and fluorescence experiments as explained below.

The anthraquinone part of Rb molecule is inherently fluorescent. Rbfluorescence is quenched in collisions with water molecules; when Rbmolecules are screened from collisions with water, theft fluorescenceincreases manifold. This phenomenon was used to study P-triblock P-105micellization [31]: As illustrated in FIG. 23, Rb fluorescence increasedsharply upon the onset of micelle formation in P-triblock solutions; ascould be expected (see, e.g.[22]), P-triblock concentrationcorresponding to the onset of micelle formation decreased withincreasing temperature.

Information produced by the fluorescent part of Rb molecule implied thatat room temperature and in 10% P-triblock solutions, about 70% of Rbmolecules were localized in the hydrophobic environment of micellecores; this is in contradiction to the information given by theparamagnetic (nitroxide) part of Rb molecule which implied that the drugwas localized in the hydrophilic environment of micelle corona.

A feasible explanation of this discrepancy is that Rb molecules werelocalized at the interface between the PPO core and PEO corona ofP-triblock micelles, hydrophobic anthraquinone part being inserted intothe micelle core while more polar nitroxide part looking into thecorona; this also indicated that in P-triblock micelles, the transitionfrom core to corona was rather sharp (within several angstroms).

Drug Localization in P-gel-nanoparticles at Room Temperature

For a 10% P-triblock/1% poly(NiPAAm) P-gel solution, similar to a 10%P-triblock solutions without a hydrogel, the EPR spectra revealed twopopulations of Rb molecules in the hydrophilic environment characterizedby the same hyperfine splitting parameters; a fraction of rotationallyrestricted molecules was slightly higher in P-gel nanoparticlesindicating that a larger fraction of drug molecules was drawn into thedepth of the nanoparticles (78% vs. 71%).

Effect of Temperature on Drug Localization in P-gel-nanoparticles

Heating a P-gel to 37° C., e.g. above gel's LCST, resulted in thechanges of the EPR parameters of Rb molecules; again, two populations ofthe drug molecules were revealed, one being characterized by n_(A)=16.3G (close to that at room temperature), and the other by n_(A)=15.7 G.The second hyperfine splitting constant was significantly lower thann_(A)=16.1 G measured at room temperature; this splitting constant wasclose to n_(A)=15.40 measured for Rb molecules localized in the bilayersof DMPC liposomes. This finding implied that heating the P-gel abovegel's LCST resulted in drug transfer into a more hydrophobicenvironment, presumably that of hydrophobic cores of P-gel nanoparticle.The overall fraction of the drug in the cores of P-gel nanoparticle was73%. These data are in agreement with those provided by the 16-DS spinprobe.

Effect of Dilution on Drug Distribution in a P-gel

Upon a 100-fold dilution of the initial 10% P-triblock/1% poly(NiPAAm)P-gel solution at room temperature, Rb was transferred from the aqueousphase into a less hydrated one, as indicated by splitting of thelow-filed line in the high-frequency EPR spectra (FIG. 11). This is asomewhat unexpected observation because drug partitioning considerationswould lead to the opposite prediction. However, three types of watermolecules should be considered for a P-gel: a free water, water inhydrogel pores, and water associated with PEO corona of nanoparticles.The degree of hydration of a hydrogel phase increases upon dilution;since Rb has a low aqueous solubility, it is drawn deeper into the lesshydrated environment. A similar effect is observed for a 16-DS probe.

An advantage is that the p-gel micelles are stable for drug delivery,but not so stable that they cannot be degraded by the body. After amatter of weeks, the stabilized p-gels will gradually destabilize. Thisallows sufficient time to function effectively as a drug deliverysystem, but the degradation will allow eventual removal from the body.This is unlike many drug delivery systems that involve stable componentsthat are slow to be removed from the body. The thermodynamics of thep-gel system direct the system toward dissolution, and instability, butthe kinetics are very slow.

Effect of Micelle Structure on the Intracellular Drug Uptake

In this study, HL-60 cells were incubated at 37° C. with DOX or Rb. Thedrugs were either dissolved in the RPMI medium (or PBS), or they weresolubilized in P-triblock PLURONIC P105 solutions of variousconcentrations.

The uptake of either drug was somewhat enhanced at P-triblockconcentration of 0.1%, which is below the CMC for the formation ofmicelles with hydrophobic cores. This is in agreement with Kabanov'sdata [33] and implies that P-triblock molecules in a unimeric form or inloose aggregates enhance the permeability of cell membranes toward thedrugs (FIG. 17) [31].

Drug sequestering in P-triblock PLURONIC P-105 micelles with hydrophobiccores caused substantial decrease in drug uptake by HL-60 cells,indicating that dense micelles inhibited drug interaction with the cells(FIG. 17).

Acoustically Activated Drug Release from Unstabilized and StabilizedP-triblock Micelles Under Continuous Wave Ultrasound

For these experiments, a home-made real-time fluorescence detectionchamber was used. (FIG. 18) The apparatus employed a single-lineargon-ion laser (Ion Laser Technology, Model 5500 A) whose beam wasdivided by a variable beam splitter (a graded metal-film neutraldensity, filter). One portion of the beam was sent directly to a siliconphotodetector (Newport Model 818-SL with 835 display) to monitor thelaser power. The other portion was directed into the glass cuvettecontaining the trial solution to be sonicated. It was designed incollaboration with Dr. Christensen (University of Utah); details of thedesign will be described elsewhere.

The effect of ultrasound on drug release from micelles was measuredbased on differences in fluorescence intensity of Rb or DOX within andoutside micelles. A constant wave and pulsed ultrasound was applied todrug solutions at 37° C. With no P-triblock present, insonation had noeffect on drug's fluorescence.

When Rb or DOX were solubilized in 10% P-triblock solution (micelleswith hydrophobic cores), insonation caused decrease in drug fluorescenceintensity.

Upon the termination of insonation, the fluorescence intensity recoveredto its original level. The effect was highly reproducible in allexperiments. The drop in fluorescence indicated that a portion of Rb orDOX was expelled from micelles under sonication. The onset of a steadyfluorescence intensity level under sonication resulted from theequilibrium between drug release and re-encapsulation.

Acoustically activated drug release was observed also forhydrogel-stabilized micelles. The reencapsulation proceeds very fast.These findings are important for the drug delivery application ofacoustically-activated micelles. In conclusion, ultrasound inducedrelease of some fraction of the encapsulated drug from micelles; drugwas re-encapsulated when ultrasound was switched off.

Acoustical Triggered Release of Drugs from Polymeric Micelles UnderPulsed Ultrasound

In clinical situation pulsed ultrasound appears to be superior to CWsince pulse, and pulse duration and sequence can be carefullycontrolled. Also, heating and burning of skin can be prevented by theapplication of pulsed ultrasound with appropriate pulse sequences.

A custom ultrasonic exposure chamber with real-time fluorescencedetection was used to measure acoustically-triggered drug release fromP-triblock PLURONIC P-105 micelles under continuous wave (CW) or pulsedultrasound in the frequency range of 20 kHz to 90 kHz. The measurementswere based on the decrease in fluorescence intensity when drug wastransferred from the micelle core to the aqueous environment. Twofluorescent drugs were used: doxorubicin (DOX) and its paramagneticanalogue, ruboxyl (Rb). P-triblock PLURONIC P-105 at variousconcentrations in aqueous solutions was used as a micelle-formingpolymer. Drug release was highest at 20 kHz ultrasound and dropped withincreasing ultrasonic frequency despite much higher power densities.These data suggest an important role of transient cavitation in drugrelease. The release of DOX was higher than that of Rb due to strongerinteraction and deeper insertion of Rb into the core of the micelles.Drug release was higher at lower P-triblock concentrations, whichpresumably results from higher local drug concentrations in the core ofP-triblock micelles when the number of micelles is low. At constantfrequency, drug release increased with increasing power density. Atconstant power density and for pulse duration longer than 0.1 s, peakrelease under pulsed ultrasound was the same as stationary release underCW ultrasound. Released drug was quickly re-encapsulated between thepulses of ultrasound, which suggests that upon leaving the sonicatedvolume, the non-extravasated and non-internalized drug would circulatein the encapsulated form, thus preventing unwanted drug interactionswith normal tissues.

Measuring Ultrasound-triggered Real-time Release of DOX and Rb fromP-triblock P-105 Micelles

The anthraquinone parts of Rb and DOX molecules are inherentlyfluorescent when excited at a wavelength of 488 nm, making themeffective as fluorescent probes. However, Rb and DOX fluorescence isquenched by collisions with water molecules (dynamic quenching). Thus,when Rb and DOX molecules are prevented from collisions with water, forinstance by their encapsulation in the hydrophobic core of micelles,their fluorescence increases two- to three-fold [72]. This feature wasused in this study to measure drug release from micelles under theaction of ultrasound.

Real-time measurements of drug release were performed using a speciallydesigned ultrasonic exposure chamber with fluorescence detection, shownschematically in FIG. 18.

Fluorescence of the drug was excited at an excitation wavelength of 488nm with an optical power of approximately 0.5 mW in a 2 mm diameterbeam. At this light intensity no photobleaching was observed, based onthe constant level of drug fluorescence during continuing irradiationfor 8 hours. The drug release was quantified by measuring the changes influorescence emissions before, during, and after the ultrasoundexposure. A fiberoptic probe (a sheathed bundle of multimode glassfibers, 3 mm entrance diameter, 0.6 numerical aperture, and 90 cm inlength) was used to collect the fluorescence emission. The light passedthrough a dielectric bandpass filter with a 35 nm bandwidth centered at535 nm (Omega Optical Model 535DF35) to a sensitive silicon detector(EG&G Model 450-1). The filter effectively cuts off emissions below 500nm, including any Rayleigh-scattered laser light. The detector signalwas digitized using a 12-bit AID converter (National Instruments) andsent, along with the digitized monitor photodetector signal, to aMacintosh computer for storage and processing. The analog outputs ofboth photodetectors were also plotted on a stripchart recorder. Thetemperature of the ultrasonic exposure chamber was maintained at 37° C.by circulating thermostated water throughout the sonicating bath.

The glass cuvette used to measure drug release had two open tubes forfilling or removing drug solution and one sealed tube in the middle toallow the excitation beam to enter the solution through a flatstationary surface. This prevents any distortions that could otherwisearise from waves on the surface of the sonicated liquid. The mainchamber of the cuvette was completely filled with the solution, and theexcess liquid partially filled the side tubes.

The experimental procedure is described below. First, fluorescenceintensity of drug in phosphate buffered saline, PBS (F_(PBS)) wasmeasured; then, without any changes in the experimental setup, the PBSsolution was carefully removed and replaced with the drug solution inP-triblock micelles. In all experiments, Rb concentration was 20 μg/mland DOX concentration was 40 μg/ml. The base fluorescence of themicellar solution (F_(mic)) was measured, after which CW or pulsedultrasound was turned on. During the “ultrasound on” phase, fluorescencedropped as shown in FIG. 19 due to drug release from the hydrophobiccore of micelles into the aqueous environment.

Digitized data were analyzed, to calculate the percentage of drugrelease from micelles. To reduce the noise, the data were Fouriertransformed, and a small magnitude narrow band noise of unknown originand its next three harmonics were filtered out. After Fourier filtering,the data were smoothed using a 10 point moving average. An example ofthe raw and filtered data is presented in FIG. 19 for DOX release from10% P-triblock micelles.

The percent of drug release was calculated assuming that F_(mic)-F_(PBS)corresponds to 100% drug release:

Release (%)=[(F_(mic)−F_(us))/(F_(mic)−F_(PBS))]×100%  (1)

where F_(us) is fluorescence during exposure to ultrasound.

The data also showed the time required to release the drug from micellesand the time required for drug re-encapsulation once the ultrasound wasturned off.

Drugs

Rb was kindly provided by Dr. Shapiro (Institute of Biochemical Physics,Moscow, Russia). DOX was supplied by the University Hospital, Universityof Utah, Salt Lake City, Utah, USA

Drug Encapsulation in P-triblock Micelles

An aliquot of Rb stock solution in 1:1 C₂H₅OH/acetone mixture wasevaporated in a vacuum evaporator; PBS or P-triblock solution in PBS wasadded to the solid Rb residue to produce a final Rb concentration of 20μg/ml. The system was vortexed for 30 s and then sonicated in asonication bath operating at 90 kHz until Rb was completely dissolved,which usually took about 15 s.

DOX was introduced into PBS or P-triblock micellar solutions from astock solution in PBS at a final concentration of 40 μg/ml , followed bya short (15 s) sonication in a sonication bath operating at 90 kHz tofacilitate drug encapsulation.

Insonation

Drug release as a function of ultrasound frequency was explored in alow-frequency range, from 20 to 90 kHz; both CW and pulsed ultrasoundwas investigated. The ultrasound power density was varied from 0 to 3W/cm² as measured by a hydrophone as described earlier [34].

The 20-kHz ultrasound was generated by a probe transducer (Sonics andMaterials, Newton, Conn.) inserted into the water bath; sonication at 47kHz was performed in a Cole-Parmer sonication bath (Cole-Parmer Inc.,Mount Vernon, Ill.); sonication at 67 and 90 kHz was performed in twodifferent Sonicor SC 100 sonication baths (Sonicor Instruments,Copaique, N.Y.). The power density was controlled by adjusting the a.c.input voltage with a Variac. The 20-kHz ultrasound probe was programmedto generate continuous wave (CW) or pulsed ultrasound of varying powerdensities and duty cycles; in the pulsed experiments both “ultrasoundon” and “ultrasound off” durations were varied. For the sonicationbaths, pulses were generated by turning the instruments on and offmanually.

Ultrasound-induced Radical Formation

Radicals produced upon collapse of transient cavitations were trappedwith 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a radical trap that formsrelatively stable adducts with hydroxyl radicals [81]. DMPO wasdissolved in PBS at a concentration of 0.1 M. The insonation wasperformed in darkness; upon the termination of insonation, an aliquot ofsolution was immediately frozen and kept in liquid nitrogen until theEPR recording.

Results and Discussion Drug Release Under the Continuous Wave and PulsedUltrasound

Using the ultrasonic exposure chamber with real-time fluorescencedetection, drug release was measured from micelles under CW or pulsedultrasound in the frequency range of 20 kHz to 90 kHz.

Examples of the release profiles of drugs from 10% P-triblock micellesare shown in FIG. 20 for Rb at 47-kHz sonication and in FIG. 21 for DOXat 20-kHz sonication respectively; both CW and pulsed ultrasound withvarious duty cycles were explored.

The drop in fluorescence intensity during the “ultrasound on” phaseindicates drug release from the hydrophobic environment of P-triblockmicelle cores into the aqueous environment, which may result either fromultrasound-induced drug diffusion out of micelles or from micelledegradation under sonication, as discussed below.

FIGS. 20 and 21 reveal fast re-encapsulation of the released drug duringthe “ultrasound off” phase of pulsed ultrasound. This is a satisfyingfinding because it suggests that upon leaving the sonicated volume, thenon-extravasated and non-internalized drug would circulate in theencapsulated form, thus preventing unwanted interactions with normaltissues. This is supported by the negligible adsorption or binding of Rbto blood proteins (albumin, fibrinogen) reported elsewhere [82].

When pulse duration was longer than 0.5 s, only negligible differenceswere observed between the magnitude of the drug release under pulsedultrasound and that under CW ultrasound (FIGS. 20 and 21).

Effect of Ultrasound Frequency

Drug release as a function of ultrasound frequency was explored in a438159.1 09032.040 U-2616 low-frequency range, using the followingtransducers: 20 kHz; 47 kHz; 67 kHz, and 90 kHz. 20 kHz ultrasound wasfound to most effective. To get comparable release at higherfrequencies, greater power densities had to be used. (See Table 3.).This is in agreement with the observations of the effect of ultrasonicfrequency on the transdermal penetration of various drugs studied byMitragotri et al. [83].

Effect of Power Density

For all frequencies studied, the release of drug increased withincreasing power density (Table 4). This was true for both stationaryrelease under CW ultrasound and peak release under pulsed ultrasound(data not shown).

Effect of Drug Lipophilicity

At the same frequencies and power densities, the release of DOX fromP-triblock micelles was noticeably higher than that of Rb (Table 4).This may be due to the deeper insersion of Rb into the interior ofP-triblock micelles reported earlier [72]. This indicates that druglipophilicity is an important factor determining the extent ofacoustically activated drug release from micelles.

Effect of p-Triblock and Drug Concentration

Not much difference was observed between Rb (or DOX) release from 10%and 1% P-triblock micelles; however, significantly higher release of Rband DOX was observed from 0.1% solutions at all frequencies and powerdensities studied. For Rb, release was between 11 to 13% from 0.1%solution vs. 5.5% from 10% solution (at 67 kHz and 2.8 W/cm²powerdensity). Data for DOX at 20 kHz are presented in FIG. 22 (note thatmeasurements of drug release from P-triblock solutions of lowconcentrations are slightly less accurate than those for 10% or 1%solutions because of decreased differences between drug fluorescence inP-triblock and PBS). Higher drug release from P-triblock solutions oflower concentrations may be due to higher local drug concentration inthe core of P-triblock micelles when the number of micelles is low, i.e.at P-triblock concentrations only slightly above the corresponding CMC(which is 0.03% for P-105 at 37° C., based on data presented in [84]).This is corroborated by the finding that for the same P-triblockconcentration of 10%, drug release indeed increased with increasinginitial concentration of drug in the solution. At a concentration of 40μg/ml, DOX release was 10%±1% (mean and s.d.), while at a concentrationof 30 μg/ml, the release was 5.5%±1% (at 67 kHz and 2.8 W/cm² powerdensity). The lower drug release at the lower drug concentration couldbe attributed to a higher ratio of PPO to DOX in the hydrophobic core ofP-triblock micelles, which favors hydrophobic interaction. It ispostulated that increased hydrophobic interaction reduces percentage ofdrug that can be released from micelle core upon the application ofultrasound. This is confirmed by the above mentioned lower release of Rbin comparison to DOX. At higher local drug concentrations in micellecores, drug/PPO hydrophobic interactions are replaced by weakerdrug/drug interactions, which facilitates drug release.

Radical Formation Under Sonication

In parallel with measuring drug release, the threshold for transientcavitation was measured by trapping radicals that were produced upon thecollapse of cavitation bubbles. Cavitation threshold increased withincreasing ultrasound frequency; at 20 kHz, radicals were observed evenat a power density as low as 0.01 W/cm², which is consistent with therelatively high efficiency of 20-kHz ultrasound for drug release frommicelles. At 67 kHz, no radicals and no drug release were observed belowa power density of 1.0 W/cm². These data are summarized in Table 5.

The data suggest that transient cavitation plays an important role intriggering drug release from micelles. It is hypothesized that shockwaves produced by transient cavitation events disrupt micelles andrelease drug into aqueous environment. During the “ultrasound off”phase, the micelles are restored and drug is re-encapsulated, whichtakes less than 1 s at 37° C.

The findings described above allow to formulate a new concept of alocalized drug delivery, based on drug encapsulating in polymericmicelles to prevent unwanted interactions with normal cells, incombination with focusing ultrasound on the tumor to enhanceintracellular drug uptake at the tumor site. Combining micellar drugdelivery with acoustic activation of micelles may be developed into anew technique of drug targeting to tumors.

Based on the results presented above, acoustically activated micellardrug delivery is believed to be an effective therapeutic technology fortargeted delivery of drugs to solid tumors.

TABLE 3 Effect of ultrasound frequency on DOX release from 10%P-triblock P-105 micelles. Frequency 20 kHz 67 kHz Power Density, 0.0330.047 0.058 0.15 1.35 1.66 2.8 W/cm² DOX Release 4.6 ± 0.5 6.5 ± 1.1 9.2± 0.56 0 5.4 ± 2.3 8.1 ± 2.1 10.7 ± 0.8 %

TABLE 4 Effect of power density on Rb and DOX release from 10%P-triblock P-105 micelles; ultrasound frequency 67 kHz. Power Density,W/cm² Drug Release (%) CW DOX Rb 1.35  5.4 ± 2.3  0.8 ± 0.25 1.66  8.1 ±2.1 3.2 ± 0.9 2.8 10.7 ± 0.8 5.5 ± 1.5

TABLE 5 Correlation between the formation of transient cavitation and Rbrelease from P-triblock micelles. Ultrasound Power Density, Radical DrugRelease from Frequency, kHz W/cm² Formation P-triblock Micelles 200.021 + Traces 0.033 + + 0.047 + + 0.058 + + 47 3.54 + + 67 0.15 − − 1.0traces − 1.35 + + 1.66 + + 2.80 + + 90 0.22 − − 0.83 − − 1.66 tracestraces

While this invention has been described with reference to certainspecific embodiments and examples, it will be recognized by thoseskilled in the art that many variations are possible without departingfrom the scope and spirit of this invention, and that the invention, asdescribed by the claims, is intended to cover all changes andmodifications of the invention which do not depart from the spirit ofthe invention.

What is claimed is:
 1. A method for stabilization of a micellecomprising molecules of a block copolymer having a hydrophobic block anda hydrophilic block where the hydrophobic block forms a core of themicelle with corona from the hydrophilic block, the method comprisingcross-linking hydrophobic blocks of the molecules without reacting oreffecting the function of hydrophilic blocks.
 2. The method of claim 1wherein the cross-linking of the core is initiated by a hydrophobicradical initiator that dissolves only in the hydrophobic blocks of thecore.
 3. A stabilized micelle comprising molecules of a block polymerhaving a hydrophobic block and a hydrophilic block where the hydrophobicblock forms a core of the micelle with corona from the hydrophilicblock, where hydrophobic blocks in the core are cross-linked withoutreacting or effecting the function of the hydrophilic block.
 4. A methodfor stabilization of a micelle formed from a block polymer having ahydrophobic block and a hydrophilic block where the hydrophobic blockforms a core of the micelle with corona from the hydrophilic block, themethod comprising introducing an amount of a hydrophobic oil in anamount effective to diffuse into the core and to stabilize the core byincreasing the hydrophobicity of the core.
 5. The method of claim 4wherein the oil is a vegetable oil.
 6. A stabilized micelle comprisingmolecules of a block polymer having a hydrophobic block and ahydrophilic block where the hydrophobic block forms a core of themicelle with corona from the hydrophilic block, with a hydrophobic oilin the hydrophobic blocks in an amount sufficient to stabilize the core.7. A method for stabilization of a micelle formed from a block polymerhaving a hydrophobic block and a hydrophilic block where the hydrophobicblock forms a core of the micelle with corona from the hydrophilicblock, the method comprising polymerizing a stimuli-responsive hydrogelin the micelle core to form an interpenetrating network in the core. 8.A method for stabilization of a micelle formed from a block polymerhaving a hydrophobic block and a hydrophilic block where the hydrophobicblock forms a core of the micelle with corona from the hydrophilicblock, the method comprising polymerizing a temperature-responsive LCSThydrogel in the micelle core to form an interpenetrating network in thecore.
 9. The method of claim 7 wherein the hydrogel is pH responsive.10. A stabilized micelle comprising molecules of a block polymer havinga hydrophobic block and a hydrophilic block where the hydrophobic blockforms a core of the micelle with corona from the hydrophilic block, thecore stabilized with a stimuli-responsive low critical solutiontemperature (LCST) hydrogel in the micelle core forming aninterpenetrating network in the core.
 11. A method for stabilization ofa micelle formed from a block polymer having a hydrophobic block and ahydrophilic block where the hydrophobic block forms a core of themicelle with corona from the hydrophilic block, the method comprisingpolymerizing a temperature-responsive low critical solution hydrogel inthe micelle core to form an interpenetrating network in the core, usingan initiator that is hydrophobic such that polymerization of hydrogeloccurs predominantly in the core, with only a minor amount occurringoutside of the core.
 12. A method for manufacture of a stabilizedmicelle delivery system for a lipophilic or hydrophilic substancecomprising: stabilizing micelles comprising a block polymer having ahydrophobic block and a hydrophilic block where the hydrophobic blockforms a core of the micelle with corona from the hydrophilic block, themethod comprising polymerizing a hydrogel with a temperature-responsivelow critical solution temperature (LCST) in the micelle core to form aninterpenetrating network in the core, using an initiator that ishydrophobic such that polymerization of hydrogel occurs in the core,with only a minor amount occurring outside of the core; controlling thetemperature of the micelles to achieve a first temperature which isbelow the LCST such that the hydrogel in the core of the micelle is in aswollen state sufficient to swell the core of the micelle; loadingsubstances into the micelles to diffuse the substance into the swollencores of the micelles, and controlling the temperature of the micellesto achieve a second temperature of the micelles above the LCST at whichthe gel collapses to a dense phase to form dense core in which thesubstance is retained in the core.
 13. The method of claim 12 whereinthe substance is a drug.
 14. The method of claim 12 wherein thetemperature-responsive hydrogel is selected from the group consisting ofN-isopropylacrylamide, N,N-diethylacrylamide N,N-diethylmethacrylamide,N-isopropylmethacrylamide, N-n-butylacrylamide, other mono- and di-alkylsubstituted acrylamides, acrylic acid, methacrylic acid, and mixturesthereof.
 15. The method of claim 12 wherein temperature responsivehydrogel is copolymerized with a monomer to change the LCST.
 16. Themethod of claim 12 wherein temperature responsive hydrogel iscopolymerized with acrylic acid to shift the LCST upward.
 17. The methodof claim 12 wherein temperature responsive hydrogel is copolymerizedwith, and the use of hydrophobic butyl acrylate to shift the LCSTdownward.
 18. The method of claim 12 wherein temperature responsivehydrogel is copolymerized with acylamide, acrylic, or methacryliccomonomer to change the LCST.
 19. The method of claim 12 whereinpolymerized hydrogel segments are formed that have a MW less than 20,000Daltons.
 20. The method of claim 12 wherein the block polymer isp-triblock.
 21. The method of claim 13 wherein the drug is released fromthe stabilized micelle by subjecting the micelle to a pulsed ultrasoundsignal.
 22. A method for administering a hydrophobic drug to a patient,the method comprising: stabilizing micelles comprising a block polymerhaving a hydrophobic block and a hydrophilic block where the hydrophobicblock forms a core of the micelle with corona from the hydrophilicblock, the method comprising polymerizing a hydro gel with atemperature-responsive low critical solution temperature (LCST) in themicelle core to form an interpenetrating network in the core, using aninitiator that is hydrophobic such that polymerization of hydrogeloccurs in the core, with only a minor amount occurring outside of thecore, controlling the temperature of the micelles to achieve a firsttemperature which is below the LCST such that the hydrogel in the coreof the micelle is in a swollen state sufficient to swell the core of themicelle, loading the drugs into the micelles to diffuse drug into theswollen cores of the micelles, controlling the temperature of themicelles to achieve a second temperature near the temperature of thebody, which temperature is above the LCST at which the gel collapses toa dense phase to form dense core in which drug is retained in the core,administering the micelles into the patient's bloodstream or desiredsite in the patient's body, and subjecting a region of the body to whichthe drug is to be administered to pulsed ultrasound signal in whichpulses are timed to release the drug when the ultrasound is on.
 23. Themethod of claim 11 wherein the temperature-responsive hydrogel isselected from the group consisting of N-isopropylacrylamide,N,N-diethylacrylamide N,N-diethylmethacrylamide,N-isopropylmethacrylamide, N-n-butylacrylamide, other mono- and di-alkylsubstituted acrylamides, acrylic acid, methacrylic acid, and mixturesthereof.
 24. The method of claim 11 wherein temperature responsivehydrogel is copolymerized with a monomer to change the LCST.
 25. Themethod of claim 11 wherein temperature responsive hydrogel iscopolymerized with acrylic acid to shift the LCST upward.
 26. The methodof claim 11 wherein temperature responsive hydrogel is copolymerizedwith, and the use of hydrophobic butyl acrylate to shift the LCSTdownward.
 27. The method of claim 11 wherein temperature responsivehydrogel is copolymerized with acylamide, acrylic, or methacryliccomonomer to change the LCST.
 28. The method of claim 11 whereinpolymerized hydrogel segments are formed that have a MW less than 20,000Daltons.
 29. The method of claim 11 wherein the block polymer isp-triblock.
 30. The method of claim 11 wherein the drug is released fromthe stabilized micelle by subjecting the micelle to a pulsed ultrasoundsignal.
 31. A method for manufacturing a stabilized micelle deliverysystem for a lipophilic or hydrophilic substance, the method comprising:stabilizing micelles comprising a block polymer having a hydrophobicblock and a hydrophilic block where the hydrophobic block forms a coreof the micelle with a corona from the hydrophilic block, saidstabilizing comprising polymerizing a hydrogel with atemperature-responsive low critical solution temperature (LCST) in themicelle core to form an interpenetrating network in the core, using aninitiator that is hydrophobic such that polymerization of the hydrogeloccurs in the core, with only a minor amount occurring outside of thecore, wherein the hydrogel is selected from the group consisting ofN-isopropylacrylamide, N,N-diethylacrylamide N,N-diethylmethacrylamide,N-isopropylmethacrylamide, N-n-butylacrylamide, other mono- and di-alkylsubstituted acrylamides, acrylic acid, methacrylic acid, and mixturesthereof; controlling the temperature of the micelles to achieve a firsttemperature which is below the LCST such that the hydrogel in the coreof the micelle is in a swollen state sufficient to swell the core of themicelle; loading substances into the micelles to diffuse the substanceinto the swollen cores of the micelles, and controlling the temperatureof the micelles to achieve a second temperature of the micelles abovethe LCST at which the gel collapses to a dense phase to form dense corein which the substance is retained in the core.
 32. A method formanufacturing a stabilized micelle delivery system for a lipophilic orhydrophilic substance, the method comprising: stabilizing micellescomprising a block polymer having a hydrophobic block and a hydrophilicblock where the hydrophobic block forms a core of the micelle with acorona from the hydrophilic block, said stabilizing comprisingpolymerizing a hydrogel with a temperature-responsive low criticalsolution temperature (LCST) in the micelle core to form aninterpenetrating network in the core, using an initiator that ishydrophobic such that polymerization of the hydrogel occurs in the core,with only a minor amount occurring outside of the core, wherein thehydrogel is copolymerized with acrylic acid to shift the LCST upward;controlling the temperature of the micelles to achieve a firsttemperature which is below the LCST such that the hydrogel in the coreof the micelle is in a swollen state sufficient to swell the core of themicelle; loading substances into the micelles to diffuse the substanceinto the swollen cores of the micelles, and controlling the temperatureof the micelles to achieve a second temperature of the micelles abovethe LCST at which the gel collapses to a dense phase to form dense corein which the substance is retained in the core.
 33. A method formanufacturing a stabilized micelle delivery system for a lipophilic orhydrophilic substance, the method comprising: stabilizing micellescomprising a block polymer having a hydrophobic block and a hydrophilicblock where the hydrophobic block forms a core of the micelle with acorona from the hydrophilic block, said stabilizing comprisingpolymerizing a hydrogel with a temperature-responsive low criticalsolution temperature (LCST) in the micelle core to form aninterpenetrating network in the core, using an initiator that ishydrophobic such that polymerization of the hydrogel occurs in the core,with only a minor amount occurring outside of the core, wherein thehydrogel is copolymerized with hydrophobic butyl acrylate to shift theLCST downward; controlling the temperature of the micelles to achieve afirst temperature which is below the LCST such that the hydrogel in thecore of the micelle is in a swollen state sufficient to swell the coreof the micelle; loading substances into the micelles to diffuse thesubstance into the swollen cores of the micelles, and controlling thetemperature of the micelles to achieve a second temperature of themicelles above the LCST at which the gel collapses to a dense phase toform dense core in which the substance is retained in the core.
 34. Amethod for manufacturing a stabilized micelle delivery system for alipophilic or hydrophilic substance, the method comprising: stabilizingmicelles comprising a block polymer having a hydrophobic block and ahydrophilic block where the hydrophobic block forms a core of themicelle with a corona from the hydrophilic block, said stabilizingcomprising polymerizing a hydrogel with a temperature-responsive lowcritical solution temperature (LCST) in the micelle core to form aninterpenetrating network in the core, using an initiator that ishydrophobic such that polymerization of the hydrogel occurs in the core,with only a minor amount occurring outside of the core, wherein thehydrogel is copolymerized with acylamide, acrylic, or methacryliccomonomer to change the LCST; controlling the temperature of themicelles to achieve a first temperature which is below the LCST suchthat the hydrogel in the core of the micelle is in a swollen statesufficient to swell the core of the micelle; loading substances into themicelles to diffuse the substance into the swollen cores of themicelles, and controlling the temperature of the micelles to achieve asecond temperature of the micelles above the LCST at which the gelcollapses to a dense phase to form dense core in which the substance isretained in the core.
 35. A method for manufacturing a stabilizedmicelle delivery system for a drug, wherein the drug is released fromthe stabilized micelle by subjecting the stabilized micelle to a pulsedultrasound signal, the method comprising: stabilizing micellescomprising a block polymer having a hydrophobic block and a hydrophilicblock where the hydrophobic block forms a core of the micelle with acorona from the hydrophilic block, said stabilizing comprisingpolymerizing a hydrogel with a temperature-responsive low criticalsolution temperature (LCST) in the micelle core to form aninterpenetrating network in the core, using an initiator that ishydrophobic such that polymerization of the hydrogel occurs in the core,with only a minor amount occurring outside of the core; controlling thetemperature of the micelles to achieve a first temperature which isbelow the LCST such that the hydrogel in the core of the micelle is in aswollen state sufficient to swell the core of the micelle; loading thedrug into the micelles to diffuse the drug into the swollen cores of themicelles; and controlling the temperature of the micelles to achieve asecond temperature of the micelles above the LCST at which the gelcollapses to a dense phase to form dense core in which the drug isretained in the core.
 36. A method for stabilizing a micelle formed froma block polymer having a hydrophobic block and a hydrophilic block wherethe hydrophobic block forms a core of the micelle with a corona from thehydrophilic block, the method comprising polymerizing atemperature-responsive low critical solution temperature (LCST) hydrogelin the micelle core to form an interpenetrating network in the core,using an initiator that is hydrophobic such that polymerization ofhydrogel occurs predominantly in the core, with only a minor amountoccurring outside of the core, wherein the hydrogel is selected from thegroup consisting of N-isopropylacrylamide, N,N-diethylacrylamideN,N-diethylmethacrylamide, N-isopropylmethacrylamide,N-n-butylacrylamide, other mono- and di-alkyl substituted acrylamides,acrylic acid, methacrylic acid, and mixtures thereof.
 37. A method forstabilizing a micelle formed from a block polymer having a hydrophobicblock and a hydrophilic block where the hydrophobic block forms a coreof the micelle with a corona from the hydrophilic block, the methodcomprising polymerizing a temperature-responsive low critical solutiontemperature (LCST) hydrogel in the micelle core to form aninterpenetrating network in the core, using an initiator that ishydrophobic such that polymerization of hydrogel occurs predominantly inthe core, with only a minor amount occurring outside of the core,wherein the hydrogel is copolymerized with hydrophobic butyl acrylate toshift the LCST downward.
 38. A method for stabilizing a micelle formedfrom a block polymer having a hydrophobic block and a hydrophilic blockwhere the hydrophobic block forms a core of the micelle with a coronafrom the hydrophilic block, the method comprising polymerizing atemperature-responsive low critical solution temperature (LCST) hydrogelin the micelle core to form an interpenetrating network in the core,using an initiator that is hydrophobic such that polymerization ofhydrogel occurs predominantly in the core, with only a minor amountoccurring outside of the core, wherein the hydrogel is copolymerizedwith acylamide, acrylic, or methacrylic comonomer to change the LCST.39. A method for stabilizing a micelle formed from a block polymerhaving a hydrophobic block and a hydrophilic block where the hydrophobicblock forms a core of the micelle with a corona from the hydrophilicblock, wherein the stabilized micelle releases a substance containedtherein in response to a pulsed ultrasound signal, the method comprisingpolymerizing a temperature-responsive low critical solution temperature(LCST) hydrogel in the micelle core to form an interpenetrating networkin the core, using an initiator that is hydrophobic such thatpolymerization of hydrogel occurs predominantly in the core, with only aminor amount occurring outside of the core.